Cytokinesis in Trypanosoma brucei, a parasitic protozoan and the causative agent of human sleeping sickness, is initiated from the anterior tip of the new flagellum attachment zone (FAZ) filament. The cytokinesis cleavage furrow ingresses uni-directionally along the longitudinal axis from the anterior towards the posterior end of the cell, without the involvement of an actomyosin contractile ring, which appeared after T. brucei diverged from the last eukaryotic common ancestor. Cytokinesis in T. brucei is known to be totally different from that in its human host, and therefore is a promising drug target. It is believed that the cytokinesis regulatory pathway in T. brucei is different from most eukaryotes and that the cleavage furrow in T. brucei involves novel components. However, little is known about the cytokinesis regulatory pathway and the cleavage furrow components in T. brucei, thus significantly hindering our understanding of the mechanisms of cytokinesis in this dreadful human pathogen. The current proposal is built upon the recently discovered cytokinesis signaling cascade, and aims to address the following questions. (1). What are the cytokinesis signaling pathways in different life cycle forms of T. brucei? We hypothesize that multiple regulators, including evolutionarily conserved protein kinases and kinetoplstid-specific regulators, cooperate at the anterior tip of the new FAZ filament to regulate cytokinesis initiation and at the cleavage furrow to promote cleavage furrow ingression. Our focus is on the mechanistic roles of two novel proteins, named CIF3 and CIF4, in cytokinesis and how they cooperate with the known cytokinesis regulators to fulfil their biological function in both the insect and bloodstream forms. (2). What are the physiological roles of protein phosphorylation and dephosphorylation in cytokinesis? The involvement of two protein kinases, TbPLK and TbAUK1, in cytokinesis suggests an extensive phosphorylation of cytokinesis regulators by the two kinases. Importantly, we identified a kinetoplastid-specific protein phosphatase, named KPP1 (Kinetoplstid-specific Protein Phosphatase 1), that appears to antagonize TbPLK. We propose to investigate the physiological roles of CIF1 and CIF2 phosphorylation by TbPLK and TbAUK1 and the contribution of KPP1 to cytokinesis. (3). What drives cleavage furrow ingression and what are the components of the cleavage furrow? We identified a novel protein that contains a kinesin motor domain and two tropomyosin domains and localizes to the cleavage furrow during cytokinesis. We thus hypothesize that T. brucei employs a novel tropomyosin-based contractile machinery for furrow ingression and a plus end-directed kinesin motor to drive the uni-directional furrow ingression from the anterior cell end (minus end of the microtubules) toward the posterior cell end (plus ends of the microtubules). The long-term goal of my laboratory is to delineate the regulatory pathway that controls cytokinesis in T. brucei and explore the mechanisms underlying the distinct cell cycle control in different life cycle forms. These studies will facilitate our fundamental understanding of the molecular basis of cytokinesis that is different from the commonly recognized cell division mechanism through the action of an actomyosin contractile ring. The outcomes from these investigations not only will have important biological significance, but also could provide novel targets for anti-trypanosome chemotherapy.